The invention relates to methods of increased protein or polypeptide expression in baculovirus vector expression systems. When recombinant-DNA techniques were developed, expectations were high regarding large-scale protein production using genetically modified bacteria. The majority of commercially attractive proteins, however, necessarily undergo post-translational modifications before they can become biologically active proteins. Hence, animal cells are now more frequently used to produce recombinant proteins.
Among animal cells, insect cells are of growing importance for the production of recombinant proteins. A convenient and versatile baculovirus vector system using insect cells has been developed. Information on the physiology of insect cells is rather scarce, however, vaccines produced via baculovirus recombinant techniques are generally well accepted. An example is the use of a baculovirus-expressed gp 160 envelope protein of human immunodeficiency virus type I as a possible AIDS vaccine in clinical trials.
Until now, large-scale production of baculovirus-expressed proteins in insect cells was limited to bioreactors of up to about 10 liters. Scale-up suspension cultures offer the best possibility. In large-scale production (see Tramper et al., Rec. Adv. Biotech., 1992, 263-284; Power and Nielsen, Cytotechnology 20: 209-219, 1996), special emphasis should be given to factors influencing cell growth and virus production. Variations in such factors greatly influence the final level of recombinant protein production.
Baculoviruses are characterized by rod-shaped virus particles which are generally occluded in occlusion bodies (also called polyhedra). The family Baculoviridae can be divided in two subfamilies: the Eubaculovirinae comprising two genera of occluded virusesxe2x80x94nuclear polyhedrosis virus (NPV) and granulosis virus (GV)xe2x80x94and the subfamily Nudobaculovirinae comprising the nonoccluded viruses. The cell and molecular biology of Autographa californica (Ac)NPV has been studied more in detail.
Many proteins have been expressed in insect cells infected with a recombinant baculovirus encoding that protein. Encoding means that such viruses are provided with a nucleic acid sequence encoding a heterologous protein and often are further provided with regulating nucleic acid sequences, such as a promoter. Most often, the polyhedrin promoter is used to express a foreign gene but the p10 promoter is equally well suited and used as well.
Several cell-lines are available for infection with recombinant baculovirus. The cell-line SF-21 was derived from ovarial tissue of the fall armyworm (Spodopterafrugiperda), A clonal isolate, SF-9, available from the American Type Culture Collection (CRL 1711), is more or less a standard cell-line for in vitro production of recombinant virus and is said to be superior in producing recombinant virus. Other cell-lines are, for example, the Hi-Five cell-line and the Tn-368 and Tn-368A cell-lines obtained from the cabbage looper (Trichoplusia ni). The most widely used media in which insect cells grow include TNM-FH, BML-TC/10, and IPL-41. These media are usually supplemented with more or less defined components, such as mammalian sera, in particular, fetal calf serum. Serum replacements have also been applied to insect-cell culture, and serum-free media, such as Ex-cell 400(trademark) and Sf900 are commercially available.
Insect cells, in general, grow on solid supports as well as in suspension, but are reported to give higher yields of virus when grown on solid supports. Infection is most efficient when cells are infected in the exponential growth phase. The amount of polyhedra and virus produced per cell, however, does not vary significantly between cells infected during different stages in the cell cycle. Cell density has a great influence on virus production. Insect cells can show a form of contact inhibition resulting in reduced virus production at higher cell densities.
The initial multiplicity of infection (xe2x80x9cm.o.i.xe2x80x9d or xe2x80x9cMOIxe2x80x9d), which is the number of infectious viruses per cell, generally influences both the fraction of infected cells and the number of polyhedra per cell at the end of infection. Optimal m.o.i. for virus production is generally considered to be at around 20-30. In a study (Licari and Bailey, Biotech. Bioeng., 37:238-246, 1991) of a recombinant baculovirus expressing xcex2-galactosidase, Sf-9 cells were infected with m.o.i. values between 0 and 100. The xcex2-galactosidase yield increased and cell density decreased with increasing m.o.i. It is generally thought that increasing or decreasing m.o.i. has only a limited affect on the maximum achievable yield of a recombinant protein per infected cell. Choosing low m.o.i., however, allows reduction of virus stock needed for infection and minimizes the risk of the generation of defective interfering particles of baculovirus. If a batch culture of insect cells is infected at high m.o.i. ( greater than 5), the ensuing infection process will be essentially synchronous, i.e., all cells will go through the infection cycle simultaneously. When cells are infected at an m.o.i.  less than 5 in a batch culture, the culture will no longer be synchronous. The culture will initially be composed of noninfected cells and cells at different points in their individual infection cycle until all cells have been infected and the production of wanted protein comes to an end. In general, in such cultures the production levels are much lower. The culture behavior is the combined behavior of the individual cells that are each in a different phase of production, thus, the suboptimal production levels. In a continuous culture, noninfected cells are added continuously and the culture will obviously be asynchronously infected.
Through designing mathematical models, it is thought possible to predict complex behaviors such as those observed when infecting cells at low m.o.i. or when propagating virus in a continuous culture system. A purely empirical analysis of the same phenomena is considered very difficult, if not impossible. At present, three models are known: the Licari and Bailey, the de Gooijer and the Power and Nielsen model. These are, despite their complexity and the effort that has gone into developing them, all first generation models, postulating about the behavior of baculoviruses expressing a model recombinant protein (xcex2-galactosidase) expressed under control of the polyhedrin promoter. They summarize, to a large extent, our present quantitative understanding of the interaction between baculovirus and insect cells, when looked upon as a black box system, with disregard to DNA and RNA accumulation and the infection cycle. The binding and initial infection processes are still quantitatively poorly understood and further work in this area is much needed.
The baculovirus expression system offers a powerful tool for recombinant protein production. Several cell-culture configurations can be used for large-scale production. These systems, however, need further optimization to take full advantage of their potential. For commercial application, large-scale and low-cost production is pivotal. Polyhedra-production systems reported in large-scale cell cultures should be dramatically improved to meet the commercial demands for a price-competitive product.
The invention provides a method for large-scale recombinant protein production using the baculovirus expression system allowing increased or improved yields of the wanted protein. The invention provides a method to produce and improve yield of a recombinant protein in insect-cell culture which comprises selecting a recombinant baculovirus encoding said protein, growing insect cells in growth medium in a culture vessel, and infecting the cells with a multiplicity of infection of  less than 0.01.
In a preferred embodiment, the invention provides a method to increase yield of a recombinant protein produced in insect-cell culture which comprises selecting a recombinant baculovirus encoding said protein, growing insect cells in growth medium in a culture vessel and infecting the cells with an inoculum of at least one baculovirus with a multiplicity of infection of  less than 0.01. Increasing yield has been a topic of several research groups. For example, Chen et al. (Drug metabolism and Disposition: 24 399-405, 1997), studied the possibility of optimizing the MOI for a co-infection approach, whereby two different baculovirus-expressed proteins were produced in insect cell culture. Contrary to the results described herein, they found for their two proteins a best MOI of approximately 0.015 to 0.03. Reducing the MOI to  less than 0.01 reduced the yield of the co-infection system of Chen et al. Radford et al. (Cytotechnology 24, 73-81, 1997), not being hindered by studying a co-infection system, clearly indicate that MOI""s  greater than 1 should always be used to maximize final process yields, again teaching against the findings of the present invention. They state that it is impossible to produce larger amounts of protein and virus per cell using low MOI and suggest adjusting the time of infection (TOI) instead.
Others, such as Nguyen et al. ( J.Biotech. 31, 205-217, 1993), do not find a solution in changing MOI, but aim at increasing yields by applying fed-batch cultures, or change the temperature under which the virus is grown (Wu et al., Cytotechnology, 9, 141-147, 1992; King et al., Biotechnol. Bioeng, 38, 1091-1099, 1991) and avoid growing the virus under MOI  less than 0.01.
These earlier results clearly differ from those provided in this description (see, for example, FIG. 1), where cultures infected with an MOI  less than 0.01 (such as 0.003, 0.001, or even 0.0001) reached higher yields than those infected with MOI 0.01 or 0.1.
A preferred embodiment of the invention provides a method to produce and improve yield of a recombinant protein in insect-cell cultures not grown in monolayer cultures. Conventional laboratory methods to produce proteins in the baculovirus expression vector system in small amounts use monolayer cultures of insect cells in culture flasks. These static cultures are normally infected with a high MOI, to ensure synchronous infection. It is pivotal that all cells are infected before the monolayer has become confluent, since contact inhibition will lead to metabolic changes in the cells which may affect the final product yield. Since it is difficult to accurately establish the cell density in monolayer cultures, it is impossible to carry out MOI experiments. It is even more useless to use a low MOI ( less than 0.01) to infect the cultures. Both over- and under-estimation of the cell density at the time of infection will lead to a significantly suboptimal protein yield.
Routinely, one uses a high MOI, which guarantees a synchronous infection of the total cell population. This implies that large virus stocks are needed to infect the monolayer culture to achieve optimal yields.
Scale-up of protein production using monolayer cultures simply means using more tissue culture flasks. The production of large amounts of protein using monolayer cultures is very labor-intensive. Furthermore, it is not possible to regulate and/or monitor important culture parameters such as dissolved oxygen concentration and pH.
The invention now provides a method suitable for all insect cell cultures and the insight that a low MOI is beneficial for optimizing yield in insect cultures other than monolayer cultures.
Different types of culture vessels can be used for culturing insect cells other than in monolayer cultures. The aim of every fermentor design is achieving sufficient aeration and high cell density, while keeping the shear forces as low as possible (Tramper et al. in xe2x80x9cRecent advantages in biotechnology,xe2x80x9d eds. Vardar-Sukan and Sukan, 1992). In the majority of the cases described in the literature, a stirred tank bioreactor equipped with a gas sparger is used. In this type of vessel, homogeneity is achieved by using an impeller. This type of fermentor can be operated in different methods. First of all is the batch method. This is the most straightforward and simple method. Cells are cultured, virus is added and the product is harvested at the end of the infection. A more complicated method is fed-batch culture. A concentrated mixture of nutrients is added to the culture vessel to achieve higher cell densities and higher volumetric product yields (Nguyen et al. 1993 Journal of Biotechnology vol. 31, p.205-217). This is more complicated since it is not always clear what the limiting nutrient is. Canceling one nutrient limitation by adding this substrate may directly lead to another substrate limitation and, thus, not necessarily to higher product yields.
A different method of mixing is used in airlift bioreactors (Wu et al. 1992 Cytotechnology vol. 9 p. 141-147). This type of vessel consists of two cylinders. The cylinder with the smallest diameter (draft tube) is placed inside the cylinder with the bigger diameter (downcommer). In the center cylinder air or another gas is sparged, creating an upward flow. At the top of the fermentor the gas leaves the fluid and the fluid goes down outside the center cylinder.
In this way, both aeration and homogeneity are achieved using sparging of gas only, due to the difference in density in the draft tube and the downcommer. This method may reduce shear stress. However, continuous aeration rates are needed to achieve proper mixing.
Another method to raise the living cell density in a fermentor is by including a spin filter or another cell-retaining device. This is called perfusion. This allows removal of waste medium from the fermentor and addition of fresh medium, while retaining the cells in the fermentor. This method results in a lot of extra equipment and more difficult fermentor operation (Caron et al. 1994 Biotechnology and Bioengineering vol. 43, p. 881-891). Furthermore, methods such as a macroporous-bed and immobilization in a gel-matrix are reported. These types of methods rely on immobilization of the cells on or inside a matrix, making it possible to remove waste medium and add fresh medium without diluting the cell culture. If cell densities, contact inhibition and other related problems of monolayer cultures would be manageable, a method according to the invention could not only be applied in the above discussed various culture systems but in monolayer cultures as well.
For example, a preferred embodiment of the invention provides a method to produce and improve yield of a recombinant protein in insect cell cultures which comprises selecting a recombinant baculovirus encoding said protein, growing the insect cells in growth medium in a culture vessel with a sufficient volume to contain at least 2 liters, and infecting the insect cells with an inoculum of at least one baculovirus with an m.o.i. of  less than 0.01 PFU of said baculovirus/cell. The invention provides a method wherein multiplicities of infection are used that are considerably lower than, for example, the m.o.i. of 1-5 leading to an asynchronously infected culture. By infecting insect cultures with a baculovirus using an m.o.i. as provided by the invention, an optimal balance is achieved between the speed of replication of the cells in relation to the speed of replication of the virus, thereby allowing optimal expression of the wanted protein. A method provided by the invention can be easily adjusted to higher or lower cell densities by adjusting the m.o.i. as well, wherein the relative ratio of virus particles available for infecting the cells in the various phases of replication remains according to the multiplicities and densities provided by the invention. A preferred embodiment of the method according to the invention comprises growing the cells in a culture vessel with a sufficient volume to contain at least 10, more preferably at least 20, and most preferably at least 50 to 250 liters growth medium, thereby allowing scaling-up of baculovirus cultures expressing heterologous proteins. One can use a culture vessel with a volume that is larger than needed for the volume of growth medium that is present, e.g., one can use 100 L culture vessels to cultivate 20-70 liters cell-culture. A preferred embodiment of the method according to the invention comprises infecting the cells at a cell density of 1xc3x97105 to 5xc3x97106 cells/ml, more preferably at 5xc3x97105 to 1,5xc3x97106 cells/ml, thereby keeping the actual volume of the virus inoculum within easily manageable limits. Yet another embodiment of the method according to the invention comprises infecting the cells with an m.o.i. ≲0.005, such as 0.003, 0.001, 0.0005 or 0.00025, whereby the inoculum is kept as small as possible. A preferred embodiment of the method according to the invention comprises selecting a recombinant baculovirus expressing the wanted protein under control of the p10 promoter. The p10 promoter is playing a role in cell lysis. Absence of the p10 protein causes the infected cells to remain intact and prevents the release of polyhedra from infected cells, thereby reducing reinfection rates but not infectivity, per se. The whole process of virus infection can now be checked visually, due to the fact that the polyhedrin gene, and thus the polyhedra, are still present. Virus infection can be observed as dense protein particles that accumulate in the cell nucleus. Another embodiment of the method according to the invention comprises growing the insect cells in a batch culture system, thereby minimizing the accumulation of defective interfering baculovirus particles which can compromise infection and replication of yet uninfected cells. Another embodiment of the method provided by the invention comprises growing the insect cells in suspension, preferably in a culture vessel such as a fermentor, which can be moderately stirred. The use of stirred suspension cultures, especially when combined with using a recombinant baculovirus wherein the wanted protein is under control of the p10 promoter to visually check virus growth (but other methods of checking, e.g., in the case of using the polyhedrin promoter, such as observing CPE, are also available) allows for a better control of the culture.
Moderate stirring of the suspension guarantees a homogenous culture in which no substrate gradients are built up and in which the cells are not subjected to too high shear forces.
Furthermore, stirring results in an efficient transfer of viruses from infected to noninfected cells, giving a higher efficiency of virus infection of cells. Since initially only about 0.1-0.3% of the cells is infected, the remaining 99.7-99.9% of cells are allowed to grow and multiply.
The invention provides a method to express and produce recombinant protein of various origin. An example provided by the invention is the production of pestivirus-derived protein to a concentration of said protein in the growth medium at harvest of at least 100, 120 or 150 xcexcg/ml, and more preferably at least 200 or even 300 xcexcg/ml. The wanted protein can also be used to prepare antigenic substances for veterinary or medical use, e.g., incorporated in a vaccine or in a diagnostic test. The wanted protein produced by a method according to the invention can, for example, be used to prepare a vaccine.
An example provided by the invention is the pestivirus E2 protein or fragments thereof, which can, for example, be used to prepare a vaccine against pestivirus infections, such as classical swine fever in pigs. In a preferred embodiment, the invention provides a vaccine comprising recombinant pestivirus E2 or Ems protein or fragments thereof characterized in that it is not being immunoaffinity purified and preferably confers protection against a pestivirus infection at the PD95 level after one single vaccination with one dose. This is particularly relevant for CSFV vaccination. When applied, CSFV vaccination generally is performed during a mass campaign in an area where an outbreak of CSFV has occurred. This calls for rapid vaccination of large numbers of animals in a relatively short period. In such a mass campaign it is of imminent importance that an adequate protection level (the number of pigs that are protected against the wild-type virus infection) is achieved rapidly. Waiting for several weeks after a first vaccination for a second vaccination in order to achieve protection greatly hampers and delays the control of the disease. Differences between various methods to produce the recombinantly expressed E2 protein, even when comparing E2 fragments expressed in baculovirus, exist. In earlier reported E2 protein production cultures, the E2 protein fragment yield varied between 20-90 xcexcg/ml (Hulst et al., J. Virol. 5435-5442, 1993; Hulst and Moormann, Cytotechnology 20:271-279, 1996), further necessitating immunoaffinity-purification with monoclonal antibodies to obtain the necessary and relevant E2 antigenic mass for single shot vaccination. Another method (using a fragment of E2 described in EP 0389034) which uses E2 harvested from the supernatant of insect cells without further immunoaffinity purification, results in an E2-based vaccine that is injected twice before a satisfying (protective) immune response is obtained. Although a vaccine (Porcilis(copyright)Pesti) comprising E2 antigen is currently registered, this vaccine needs to be applied twice, thereby seriously hampering the usefulness of vaccinating against classical swine fever infections with this vaccine since it takes at least two vaccinations with a 4-week interval to provide the wanted immune response.
These problems (which are solved by the present invention), among others, relate to a low concentration of the relevant antigenic substance, in this case the E2 protein fragments in the starting material, e.g., the cell culture supernatant, from which the vaccine is prepared. In theory, one can further accumulate antigenic mass by purification and condensation methods known in the art. However, this does not lead to a commercially attractive vaccine production because of high costs per dose. Another example is the pestivirus Ems protein, which can also be used in a vaccine, in diagnostic tests or in other therapeutic substances.
For example, Ruggli et al. (Virus Genes 10:115-126, 1995) grows a baculovirus expressing E2 in monolayer insect cell culture to a maximum yield of no more than 5-10 xcexcg/106 cells. Furthermore, Moser et al. (Vet. Microbiol. 51:41-53), grows the E2 of Ruggli et al., in monolayer insect cell culture using an MOI of 5 and cannot produce enough antigen in unconcentrated form for ELISA purposes. In their experience, further purification by nickel-chelate affinity chromatography of the protein is a prerequisite to simplify handling and improve ELISA quality. No vaccine preparation was contemplated with the thus prepared E2 protein.
When vaccination was the aim of the research, it was found that vaccination needed to occur twice, using an immunopurified E2 protein, to achieve a certain measure of protection. For example, in Hulst et al. (J. Virol. 67:5435-5442, 1993), WC 95/35380, and van Rijn et al. (J. Gen. Virol. 77:2737-2745, 1996), E2 was produced in monolayers and immunoaffinity purified to achieve a protective vaccine.
An example provided by the invention is a vaccine comprising recombinant CSFV E2 protein fragments which, now that sufficient large amounts can be produced, no longer need to be immunoaffinity purified before it is incorporated in a vaccine that confers protection (at a protective dose level of 95% (PD95)) against a classical swine fever virus infection within two to three weeks after the animals received one single vaccination with one dose. A method provided by the invention provides a vaccine comprising recombinant pestivirus E2 or Ems protein, or fragments thereof, that confers protection against a pestivirus infection after one single vaccination with one dose, while the protein fragment has not been purified by immunoaffinity. The invention also provides a vaccine comprising a protein provided by the invention which additionally comprises an adjuvant. Suitable adjuvants are known to the average person skilled in the art, e.g., Freund adjuvants, or aluminum hydroxide, or emulsions, such as water-in-oil, double water-in-oil, or oil-in-water emulsions. The desired protein can also be used to prepare other substances for veterinary or medical use. Yet another example provided by the invention is a hormone-like substance, such as the follicle stimulating hormone FSH (xcex1-units and/or xcex2-units and complexes and fragments thereof), which can be produced by infecting an insect cell culture with one baculovirus expressing the xcex1-unit and/or with another baculovirus expressing the xcex2-unit in the culture. A method according to the invention can also be used for large-scale and low-cost production of recombinant baculoviruses as bioinsecticides. A preferred embodiment is the use of recombinant viruses utilizing the p10 promoter for foreign gene expression in the production of bioinsecticides since insects generally get less infected by baculovirus lacking the polyhedra gene. Culturing other recombinant baculoviruses expressing other recombinant proteins with a method according to the invention and production and/or use of such virus proteins for incorporation in insecticidal, medical, therapeutic, and/or antigenic substances or products is within the skills of the artisan. The invention is further illustrated in the following experiments but is not limited thereto.